The structured communities of environmental bacteria known as biofilms are pervasive in wet environments. Biofilms can be essential components in natural processes but can also be disruptive to industrial systems, be the source of harmful medical and dental health issues, cause environmental problems such as transporting invasive species, and decrease the fuel efficiency of ocean going vessels. The ability to measure biofilms is essential for understanding and addressing associated issues. When fully hydrated, the bulk properties of biofilms can be very similar to those of water, making it difficult to delineate the barrier between the biofilm and the surrounding bulk liquid.
Reliable and repeatable methods to observe biofilm structure and properties in three dimensions are needed in order to improve our fundamental understanding and to provide a platform to study new materials and treatments that may be used to prevent biofilm growth. Historically, microscopy has played an important role in the discovery and understanding of microbes. In situ imaging techniques are highly desirable because biofilms can vary significantly from sample to sample. Statistical averages from repeated experiments offer poor measurement resolution and large standard deviations. If instead a biofilm could be investigated non-destructively, its structure could be observed over time—during colonization or while an intervention is applied, for example.
New chemicals and drugs are needed to mediate the negative effects of biofilms. However, non-destructive tools to screen new treatments lack key abilities to study biofilms with high spatial and temporal resolution.
A common technique for 3D imaging of biological samples is confocal scanning laser microscopy (CLSM) in combination with fluorescent staining. This type of microscopy has the advantage of providing axial (vertical) information in addition to lateral (horizontal). This is accomplished by collecting data from a scanned array of points in a series of vertically separated planes. Confocal microscopy can be used to reconstruct 3D images of bacterial biofilms. Observing the “edge” or “surface” of the biofilm is not done directly; rather, the data cube is analyzed and a threshold is algorithmically determined to estimate the position of the edge. There are two major limitations of this approach with respect to the ability to use this data to study surface topology of biofilms. Namely, confocal microscopy is necessarily limited to a relatively small field of view (especially if high axial or lateral resolution is desired) and the indirect nature of the surface measurement adds uncertainty to the measurement.
Similarly, electron microscopy (scanning or transmission) and atomic force microscopy (AFM) are limited by relatively small field of view and typically require exposure of the sample to destructive environments. In the case of SEM or TEM, bacteria must also be killed, fixed, desiccated and exposed to high vacuum. In AFM, it is possible to measure wet samples, though desiccation is required for optimal resolution, and contact with a stylus is always required.
Optical techniques for studying biofilms are of particular interest because they are non-destructive and capable of real-time or near real-time in situ measurements. Light and epifluorescence microscopy are often paired with specific staining of microbiological samples. While simple and fast, light microscopy is also limited by low resolution and narrow depth of field. Low resolution is particularly apparent in the axial (vertical) dimension because it is constrained by the depth of field of the objective lens.
The field of view that can be seen by common biofilm imaging techniques narrows as higher magnification lenses are used to increase resolving power of the system. The high resolution necessary to resolve individual bacteria (>˜400× magnification) may limit the field of view (<450 μm) such that it can be difficult to observe medium or large scale features (e.g., thickness and surface roughness) that are key features in understanding biofilms. Often, the high magnification needed to image individual cells requires very low working distance or even contact with the biofilm, which fundamentally alters the biofilm's natural structure.
Optical coherence tomography (OCT) is a prominent 3-D biomedical imaging technique that is based on low-coherence interferometry. OCT typically employs near-infrared light that can penetrate shallowly into soft tissue samples. It is similar in function to ultrasound imaging, but has higher resolution owing to the use of a broad band light source. Light reflected from near surface features of a sample interferes with light directed at a reference arm. However, OCT is limited to micrometer vertical resolution by the long wavelength of the light source. OCT is well-suited for applications where light must penetrate the surface such as in ophthalmology.
White light interferometry (WLI) is a specialized technique designed to measure surface profiles over large areas with high precision. WLI is typically used for surface metrology of solids. The technique is similar to OCT in that it is based on low-coherence interferometry. However, in WLI light reflects from the sample surface and does not penetrate it because low wavelength light is used rather than near-infrared. WLI instruments are designed to measure the 3-dimensional profile of a relatively large area of a sample. Lateral resolution is unchanged from traditional light microscopy (i.e. limited by the diffraction) but the vertical resolution is extremely fine.
A comparison of microscopic imaging techniques is shown in Table 1. The table compares lateral resolution, vertical resolution and field of view for each type of microscopy. WLI is unique in this list owing to the extremely fine vertical resolution and the ability to maintain this high resolution when imaging a large field of view. WLI has potential to vastly improve both the vertical resolution and the size of a biofilm sample that can be imaged in a single field of view.
TABLE 1Resolution and field of view of common biofilm imaging techniquesLateralVerticalField ofMethodresolution ResolutionViewNotesWhite light3.7 μm3 nm2-6 mmReflective surface requiredinterferometryOptical~400 nm~400 nm<450 μmNarrow depth of field andmicroscopysmall field of viewOptical Coherence 4 μm3 μm2.9 mmLow resolutionTomographyConfocal~250 nm3 nm<450 μmObtaining large field ofmicroscopylimitview requiresscanning/stitchingCLSM~150 nm400 nm<450 μmThin sample required,limitsmall field of view, poorvertical resolutionFluorescence~250 nm400 nm<450 μmSample must bemicroscopylimitfluorescent or labeledFluorescence~30 nm<100 nm1-2 μmSample must besuper-resolutionfluorescent or labeledmicroscopyPhotoacoustic100-200μm10 μm150 μmSample exposed to air, lowspectroscopyresolutionUltrasonic~50 μm15 μm>1 mmCan't resolve low acousticimaging(scanning)impedance differencesAFM (liquid)<10 nm<1 nm~10 μmRequires contact, weaklybound samples lowerresolution, small field ofviewEnvironmental1-20 nm~1nm~12 μmDestructive sampleSEMforpreparationhigh resolution
WLI is capable of high vertical resolution because it uses interferometry to measure the distance from the objective lens to the sample. In a typical WLI microscope a source of white light (e.g., a light emitting diode) is aimed at a beam splitter. Part of the beam is directed to the sample while the remainder is reflected off of a smooth reference mirror. The reflected light from these two surfaces recombine on the imaging detector and create interference fringes when the optical path length of the experimental and reference beams are nearly equal. In some interferometric objective lenses the mirror is positioned differently than the standard Michelson-type configuration (called a Mirau objective), but the principle and the effect are the same.
When operated in vertical scanning mode, the objective is lowered through a user-defined vertical range. Fringes of light intensity appear in the image whenever a reflective surface in the sample is at an optical path distance equal to the distance to the reference mirror. The time at which fringes appear during the vertical scan indicates the height of the sample surface features. With WLI, vertical resolution far exceeds the Rayleigh criterion because it is easy to observe small differences in optical path through subtle shifts in the interference fringes. In vertical scanning mode differences in vertical height as low as 3 nm can be observed. WLI is does not suffer 2π ambiguity that occurs with high coherence sources like lasers. The short fringe packet produced by white light can be assigned to an absolute height value.
WLI has rarely been used to measure soft surfaces of living organisms or cells or specifically for biofilms. The primary reason for this is that biofilms grow in aqueous environments. Water complicates WLI measurements because its increased refractive index relative to air leads to a shift in optical path length of light passing through it. Biofilm has a refractive index that is almost equal to water because much of a biofilm's volume is composed of water. As a result, a biofilm's surface will generally not reflect light. To resolve a surface or interface on an interferometric optical microscope the surface must be at least partially reflective. Non-reflective surfaces will not return enough light back to the microscope objective to generate measurable fringe contrast. For the same reason, samples or sample features with steep slopes will often not reflect in the direction of the objective lens and are not measurable. In these cases, the resulting profile image will have points or regions of missing or distorted data.
A limited number of previous studies have used WLI to measure the interface of wet biological samples. In these studies optical profiles were often collected from wet samples that were exposed to open air. Doing so for biofilms is not desirable because evaporation from the surface of the biofilm can significantly change its structure. In fact, evaporation can make imaging the liquid layer challenging because the interface must be stable and free of vibration/movement to produce interference in the reflected light.
WLI has also been used to measure the presence of cells by observing the shift they cause in optical path length. The presence of cells that are semi-transparent and of slightly different density than the surrounding water may lead to a slight change in refractive index and thus a slight change in optical path length. In this case, it is only possible to observe the presence of cells and their relative thickness. Absolute measurement of thickness is only possible if the refractive index of the cells is known in advance.
Measuring the thickness of a biofilm on a surface provides a means of assessing how much biomass has accumulated: an important factor to consider when testing and evaluating strategies meant to inhibit biofilm formation. Unlike other methods (e.g., weight gain or extraction and determination of total organic carbon), WLI microscopy would allow a materials scientist to determine precisely where on a surface biomass is accumulating. For example, a corrosion engineer may want to examine if biofilms are forming in locations where crack, crevice, or galvanic corrosion occurs. Thickness is also roughly associated with the potential for anoxia at the base of the biofilm and with the diffusion of solutes into or out of a biofilm. The thickness of a biofilm is also critical to the function of percolating filters, membrane biofilm reactors, and other biofilm dependent strategies used in wastewater remediation. Biofilm cohesion and polysaccharide concentration increase with depth, not age in biofilms.
Additionally, the surface texture (roughness) of biofilms has an impact on fluid dynamics and with drag in particular. Topology and its effects on fluid dynamics also influence the diffusion of oxygen to the base of a biofilm. When studying biofilm growth on a textured surface, e.g., microtopography used in an attempt to alter hydrodynamics or to prevent biofouling, it may be informative to see whether the surface of the film follows the underlying topography of the substrate or if any other patterns of heterogeneity in the film are observed that correspond with preexisting patterns made in the substrate.
As described above there are many microscopy methods that can be used to study biofilms. Each method may also have a specific apparatus that positions the sample and provides suitable optical properties for imaging. Apparatuses that are designed for live biofilms must also support bacterial growth. No apparatus exists that can position a biofilm for WLI imaging and provide suitable optical conditions.
Two of the most common apparatuses for biofilm imaging are multi-well plates and flow cells. A multi-well plate is a flat plate with many “wells” that each can be used as small test tubes. Multi-well plates are a standard tool in analytical research and in diagnostic laboratories. Multi-well plates are also known as microplates or microtiter plates. Each plate may have 6, 12, 24, 48, 96, 384 or 1536 sample wells. The wells are arranged in a rectangle and are typically molded into a 5 inch by 3.33 inch plastic rectangle. The dimensions of the plate and each well are standardized for interoperability. Numerous instruments can be used to automatically fill each well with a desired analyte and complete analysis. A large market exists for robots that handle, transfer, store, analyze and clean multi-well plates. A common use for multi-well plates is in the enzyme-linked immunosorbent assay (ELISA), which is frequently used in medical diagnostic testing.
Specialty multi-well plates are manufactured for a specific assay or analytical method. Today, there are multi-well plates for many applications in life science research. There are plates for filtration, separation, storage, reaction mixing, detection of antimicrobial activity, cell culture, and optical detection. For example, multi-well plates are made with a flat glass bottom that is suited to microscopy or spectroscopy. Typically, when used for microscopy, the plate is placed bottom down on a microscope stage. The microscope must function in inverted configuration and “look up” at the bottom of each well.
Multi-well plates have been designed to study biofilms and antimicrobials that target biofilms. The plates used for this purpose can follow the Minimum Biofilm Eradication Concentration (MBEC) protocol and are referred to as MBEC plates or as a Calgary Biofilm Device. MBEC plates have a special lid with pegs designed to be partially submerged in wells that are inoculated with bacterial culture. The pegs then become coated in biofilm and can be removed for further analysis.
Flow cells are tools that are designed to study biofilms in dynamic conditions. Biofilms in the environment may encounter flowing liquid, depletion of nutrients, or toxic conditions. Flow cells provide an environment in which these variables and many others can be controlled and monitored. Flow cells typically have an experimental chamber with an inlet and an outlet. Liquid can enter and pass through the experimental chamber. Pumps are used to direct flow of liquid in these systems. When the flow cell is designed for microscopy the experimental chamber has a viewing window. The viewing window must have correct positioning and optical properties for the chosen microscopy technique.
What is needed to advance the study and prevention of bacterial biofilms is an apparatus and method for non-destructive, high resolution measurement and monitoring of biofilm thickness and topology.